This invention relates the fields of recombinant DNA technology, transgenic animals and signal transduction. More specifically, a transgenic nonhuman animal is provided wherein the osteopontin gene has been altered. Methods of using such animals to assess osteopontin""s role in the modulation of cellular activities are also provided.
Several publications are referenced in this application by superscript numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Osteopontin (OPN) is a secreted phosphoprotein found in the collagenous extracellular matrix of mineralized tissues and in many body fluids, notably plasma, urine, bile and milk.(1-3) The protein has a GRGDS integrin-binding sequence that interacts with integrins of the xcex1v class, and it can facilitate attachment of cells to various surfaces, for example during the attachment of osteoclasts to bone.(4,5) Sequence motifs in OPN that have been well conserved among avian and mammalian species include the RGD sequence just N-terminal to a thrombin cleavage site, an Asp-rich sequence with possible importance in binding to calcified tissues, a C-terminal heparin-binding domain, and multiple serine residues in contexts appropriate for phosphorylation by casein kinase II or mammary gland kinase.(6) The synthesis of OPN is induced when T cells are activated,(7) when JB6 epidermal cells are treated with 12-O-tetradecanoyl-phorbol-13-acetate(8) and when Ras becomes activated and cells acquire a metastatic phenotype.(9) Indeed, various experiments have shown that OPN is involved in the metastatic process.(10-12) 
In addition to a cell attachment capability, OPN has properties of a cytokine.(7) For example, it can activate c-src and stimulate phosphoinositide 3-kinase activity in target cells.(13,14) OPN can inhibit the induction by lipopolysaccharide and xcex3-interferon of inducible nitric oxide synthase (iNOS, type II nitric oxide synthase).(15) This inhibition of iNOS transcription correlates with the ability of OPN to protect tumor cells from being killed by activated macrophages,(16,17) suggesting that perhaps this is how OPN contributes to the metastatic phenotype. Osteopontin is produced at high levels by the macrophages found in granulomas of diverse etiology, including those induced by Mycobacterium tuberculosis,(19,20) consistent with its having an anti-inflammatory role. An anti-infectious role has long been suspected because of its association with resistance to certain infectious agents.(7) OPN also induces cellular chemotaxis and haptotaxis,(21,22) and it stimulates the infiltration of monocytes and macrophages to sites of subcutaneous OPN injection,(23) possibly through a mechanism involving CD44.(24) There is a strong association between enhanced OPN expression and monocyte/macrophage infiltration at sites of focal injury in the kidney.(25-27) 
Despite the variety of activities attributed to OPN, and its prominence in many normal and pathological tissues, its significance to the vertebrate organism remains to be elucidated. It is frequently found in pathological calcifications such as atherosclerotic plaques,(2) sclerotic glomeruli,(28) and kidney stones.(29,30) Its high expression in osteogenic cells and its accumulation in the calcified extracellular matrices of bone and teeth have been well established, seemingly implicating OPN in the development and remodeling processes of mineralized tissues.(3) 
Its presence at mineralized tissue surfaces and interfaces(31) and its facilitation of phagocytosis of OPN-coated particulates are consistent with a role in promoting cell attachment and removal of foreign bodies.(32) Its prominent distribution throughout bone, and in particular its concentration at cement lines, has prompted the suggestion that OPN participates in hard tissue cohesion and may promote interfacial adhesion between apposing substrata.(31,33) Other in vitro studies have identified OPN as a potent inhibitor of hydroxyapatite (calcium phosphate) crystal formation and growth. (33,34). 
The precise roles of osteopontin in normal tissue development and maintenance, as well as in embryogenesis and fetal development are not known at this time. Due to the putative biological importance of osteopontin in bone formation and cell attachment, the osteopontin gene is an important target for embryonic stem cell manipulation.
The generation of osteopontin deficient-transgenic mice would aid in defining the normal role(s) of osteopontin and facilitate the use of an animal model of osteopontin deficiency in the design and assessment of chemical approaches to inhibiting or augmenting osteopontin activity. Such osteopontin modified transgenic mice may also be as a source of cells for cell culture.
This invention provides non-human transgenic animals in which the osteopontin gene has been altered and methods of use thereof. The osteopontin knockout mice of the invention are fertile and develop normally.
Osteopontin plays a role in numerous physiological processes. Osteopontin-related processes include, but are not limited to, bone remodeling, angiogenesis, inhibition of nitric oxide production, renal pathologies, atherosclerosis, monocyte differentiation, osteoporosis and osteoclast function. However, the molecular mechanisms by which osteopontin effectuates these processes have yet to be elucidated.
In a preferred embodiment of the invention, mice transgenic for the osteopontin gene are provided. Such mice may be used to advantage in assays for the identification of therapeutic agents useful for the treatment of osteopontin related pathologies.
In accordance with one aspect of the present invention, it has been discovered that osteopontin knockout mice are resistant to ovariectomized-induced osteoporosis. Thus, these mice may be used to advantage to screen therapeutic agents that inhibit or promote osteoporosis.
In yet another aspect of the invention, it has been discovered that osteopontin-deficient mice are more susceptible to ischemic damage of the kidney than are wild-type mice. Accordingly, methods are provided for assessing therapeutic agents for the treatment such renal disorders.
Osteopontin is a highly conserved plasma protein. While antibodies to the protein exist, antibodies specific for all of the epitopes on the protein are difficult to obtain as these highly conserved regions will not be recognized as xe2x80x9cnon-selfxe2x80x9d following antigenic stimulation. The osteopontin knock-out mice of the invention are used in methods for the development of osteopontin-specific monoclonal antibodies. Use of the knock out mice described herein should provide a superior array of antibodies specific for osteopontin.